Translocation of the HIV-1 pre-integration complex (PIC) into the nucleus of an infected cell is a critical step in viral replication. During the previous funding period of this grant, we demonstrated that arylene bis(methylketone) compounds effectively and selectively inhibit HIV-1 nuclear translocation and replication of the virus in primary cells. We also demonstrated that the molecular mechanism of action of these compounds involves drug interaction with both reverse transcriptase (RT) and matrix (MA) proteins. We initiated studies of viral resistance to these compounds and obtained partially resistant isolate. We now propose to clone and genetically characterize this isolate. identification of mutations responsible for resistance to arylene bis(methylketone) compounds would validate the proposed mechanism of their activity and help evaluate their usefulness for future clinical applications as an addition to multi-drug cocktail. In an attempt to eliminate dependence of nuclear import inhibitors on interaction with RT, we designed a novel class of naphthoquinone compounds. Preliminary results demonstrated that some of these compounds specifically inhibited HIV-1 nuclear import in vitro and blocked viral replication in macrophage cultures without cytotoxic effects. We propose studies to determine the molecular mechanism of action of naphthoquinone compounds. We also expect these new compounds to be active against the virus resistant to arylene bis(methylketone) drugs. Studies by our and other groups demonstrated the important role of Vpr in HIV-I nuclear import and replication in macrophages. This Vpr function depends on its interaction with the cellular nuclear import factor, karyopherin. Based on our preliminary results that identify interacting sites on Vpr and karyopherin molecules, we propose to design and test compounds that will inhibit interaction between these proteins. Upon completion, these studies are expected to define potent anti-HIV compounds working through a novel mechanism different from that of any other currently used drug.